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Fluorescence assay for kinase activity

a kinase activity and fluorescence assay technology, applied in the field of fluorescence assay for kinase activity, can solve the problems of affecting the recognition of kinases by certain kinases, affecting the reactivity of certain kinases, and few examples of sensors capable of such assays

Inactive Publication Date: 2005-06-14
MASSACHUSETTS INST OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides new amino acid residues that can bind to Mg2+ and enhance their fluorescence. These residues can be used to create peptides and sensors that can detect the presence of a specific kinase. The amino acid residues are located near a hydroxyl amino acid that can be phosphorylated by the kinase. The new residues can be separated from the hydroxyl amino acid by a peptide that can form a β-turn conformation. This invention allows for the creation of more effective tools for studying metal binding and protein-protein interactions.

Problems solved by technology

Currently, few examples of sensors capable of such assays exist.
However, these types of probes, with fluorophores adjacent to the phosphorylated residue or very large fluorophores may interfere with their recognition by and reactivity with certain kinases.

Method used

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  • Fluorescence assay for kinase activity
  • Fluorescence assay for kinase activity
  • Fluorescence assay for kinase activity

Examples

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Peptide Synthesis:

[0045]Peptides were synthesized using standard Fmoc amino acid protection chemistry on Fmoc-PAL-PEG-PS resin (0.22 mmol equiv.). Couplings of Fmoc-protected amino acids to the resin were carried out with 1-benzotriazolyoxytris (pyrrolidino) phophonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (H.OBt) and diisopropylethylamine (DIEA) or O-(7-azabenzotrazol-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HATU) and DIEA to generate the activated ester. The resin was swelled in dichloromethane (5 min.) then DMF (5 min.) prior to synthesis. All amino acids other than the Sox, phosphoserine, phosphothreonine and phosphotyrosine were added by the following representative procedure: removal of the Fmoc group (20% piperidine solution in DMF, 3×5 min.), wash (DMF, 5×1 min.), coupling (amino acid / PyBOP / DIEA, 6:6:6, 0.05 M in DMF, 45 min.), rinse (DMF, 2×1 min; DCM, 2×1 min.). To couple the Sox residue, double coupling with 2 equivalents each time was used (Fm...

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Abstract

The present invention provides a sensor and methods for determining kinase activity.

Description

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]The subject matter of this application was developed in part with funding from the National Institute of Health (grant application no. GM64346). The government may have certain rights in this technology.BACKGROUND[0002]The present invention provides sensors to monitor protein kinase activity continuously with a fluorescence readout. The sensor requires minimal perturbation of a protein kinase peptide substrate. The fluorescence response with respect to time over the course of the reaction corresponds to enzyme activity. The sensor of the present invention can be used in high-throughput screening of inhibitors or substrates, detection of activity in cell extracts or enzyme purifications, spatial or temporal localization of kinase activity in a cell, and elucidation of complicated signal transduction pathways.[0003]Protein kinases are involved in all aspects of regulation within cells. A protein kinase catalyzes the transfer of a phosph...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07D215/00C07D215/12C07K14/00C07D215/26C07K2/00C07K7/06G01N33/68
CPCC07K7/06C07D215/26
Inventor IMPERIALI, BARBARASHULTS, MELISSA D.
Owner MASSACHUSETTS INST OF TECH
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